FIELD OF THE INVENTION
[0001] The present invention relates to a novel fat emulsion comprising as a main ingredient
a polyene antifungal antibiotic.
BACKGROUND OF THE INVENTION
[0002] About 30 years have passed since polyene antifungal antibiotics represented by Amphotericin
B were developed; even now, polyene antifungal antibiotics are useful as important
antifungal agents which can be generally administered and by which reliable effects
can expect.
[0003] However, their clinical use is extremely limited by serious side effects such as
hemolytic toxicity and nephrotoxicity and hence, these antifungal agents encounter
a problem that chemotherapy does not work satisfactorily.
[0004] In addition, when polyene antifungal antibiotics are applied in the form of injection,
a surface active agent called sodium deoxycholate which is irritative and hemolytic
must be used and an improvement in medical preparation has been desired.
[0005] In recent years, in order to alleviate these side effects, polyene antifungal antibiotics
have been administered either as liposome preparations comprising phospholipids or
as fat emulsion preparations obtained by emulsifying soybean oil with a small quantity
of phospholipid (Szoka, F.C. Jr., et al., Antimicrobial Agents and Chemotherapy, 31,
421-429, 1987 [hereafter referred to as "Publication No. 1"], Kirsh, R. et al., Journal
of Infectious Diseases, 158, 1065-1070, 1988 [hereafter referred to as
"Publication No. 2"], Japanese Patent Application Laid-Open No. 66123/89 [hereafter
referred to as "Publication No. 3"].
[0006] However, these various liposome preparations and fat emulsion preparations involve
a serious defect that the preparations are hardly effective for alleviating nephrotoxicity
which is the most serious problem in clinical use, although these preparations successfully
reduce hemolytic toxicity possessed by the polyene antifungal antibiotics and alleviate
acute toxicity.
[0007] In addition, these various liposome preparations and fat emulsion preparations are
characterized by undergoing phagocytosis with macrophage, etc. gathered at the infected
site. However, in evaluation on a general level, a defect encounters that most of
the drug administered undergo phagocytosis by reticuloendothelial cells represented
by liver and spleen to migrate and true transfer of the drug into the infected site
is not necessarily effective.
[0008] On the other hand, in view of manufacturing medical preparations and safety of the
preparations, liposome preparations are questionable in production in an industrial
scale. The liposome preparations also involve a serious problem in stability during
storage because of increase in particle size due to aggregation.
[0009] Fat emulsions which have been hitherto used clinically as nutrient supplementation
fluid have been used as injection preparations of various drugs, and utility of these
preparations is known. However, it was difficult to apply fat emulsion preparations
to polyene antifungal antibiotics because such preparations encounter serious problems
in preparing emulsions and in stability of the preparations due to amphiphatic property
of the drugs and poor solubility in soybean oil, etc. It was thus difficult to overcome
the problem.
[0010] In general, an administered drug migrates and is distributed in the body depending
on the property inherent possessed by the drug molecule. When a part of the drug reaches
the site to be acted, its pharmaceutical effect is exhibited. In this case, it is
preferred that the drug be concentrated only at the site necessary for exhibiting
the pharmaceutical effect. In general, however, a drug is widely distributed over
the entire body and migrates also to the sites which do not require the drug; this
sometimes causes side effects. Therefore, it is important and necessary to improve
a disposition of a drug in the body.
[0011] In view of the situations described above, the present inventors have made extensive
investigations on preparation forms for administration which can reduce the hemolytic
toxicity and nephrotoxicity and facilitate excellent migration of a drug toward infected
sites without adversely affecting the mechanism itself of pharmacological action (antifungal
action) of polyene antifungal antibiotic on a molecular level. As a result, the present
inventors have finally reached the present invention.
[0012] An object of the present invention lies in reducing nephrotoxicity of polyene antifungal
antibiotics which is definitely the most serious problem in clinical field and providing
medical preparations which enable to administering an effective dose of a drug safely
without any concern of inducing a serious nephrotic disturbance.
DISCLOSURE OF INVENTION
[0013] A characteristic feature of the present invention resides in limiting a relative
proportion of each ingredient of a polyene antifungal antibiotic, a simple lipid,
phospholipid and water in the composition, upon preparing a fat emulsion comprising
the polyene antifungal antibiotic as a main ingredient.
[0014] In the present invention, the polyene antifungal antibiotic is incorporated in an
amount of 0.005 to 5% (w/v) based on the total weight of the fat emulsion.
[0015] In the present invention, the simple lipid is incorporated in an amount of 0.5 to
30% (w/v) based on the total weight of the fat emulsion.
[0016] In the present invention, the phospholipid is incorporated in an amount of 0.15 to
2 times in a weight ratio based on the simple lipid described above.
[0017] Water which is the ingredient of the present invention is incorporated in a suitable
amount.
[0018] The fat emulsion of the present invention is characterized in its ingredients and
proportion of the ingredient, as compared to conventionally known liposome preparations
or fat emulsions. That is, the fat emulsion of the present invention is greatly different
from liposome in the construction and structure in that the emulsion is obtained by
emulsifying a simple lipid such as soybean oil, etc. with phospholipid. Furthermore,
its phospholipid content is greatly different from conventional fat emulsions used
as preparation forms of various drugs for intravenous administration.
[0019] By the above construction, the effects that could not be obtained by conventional
liposome preparations and fat emulsions can be obtained. Hereafter the fat emulsion
is described in detail.
[0020] The fat emulsion of the present invention does not contain emulsion particles of
1 µm or greater.
[0021] A mean particle diameter of the fat emulsion of the present invention is in the range
of from 10 nm, inclusive, to less than 200 nm. This is because the emulsion particles
readily migrate from blood vessel into the focus tissue at the site where vascular
permeability is accentuated due to inflammatory reaction caused by infection with
fungi, etc.
[0022] At such infected sites, many emulsion particles of the present invention selectively
migrate from blood vessel into the focus tissue.
At the same time, the drug included in the emulsion particle also migrates into the
focus. By such migration, the drug migrates selectively into the focus easily so that
the drug concentration at the focus site increases, whereby the effect of the drug
can be enhanced.
[0023] A mean particle diameter of the fat emulsion in accordance with the present invention
is preferably 100 nm or less. The fat emulsion having such a diameter range is excellent
in avoiding intake by the reticuloendothelial system.
[0024] In addition, where the fat emulsion of the present invention is administered, the
disturbance noted with the polyene antifungal antibiotic against the kidney function
is not recognized at all. When the fat emulsion of the present invention is applied,
an amount of the polyene antifungal antibiotic migrated into kidney can be extremely
minimized and as the result, it is considered that alleviation of kidney impairment
can be achieved.
[0025] Another characteristic feature of the present invention resides in using finely divided,
stable emulsion particles as a preparation form of the polyene antifungal antibiotic.
[0026] By dividing the antibiotic into fine particles, not only the effect described above
but also the effect of maintaining blood concentration of the drug can be obtained
by inhibiting non-specific uptake of the drug by the reticuloendothelial tissue or
the like.
[0027] The polyene antifungal antibiotic is a relatively unstable drug and it is known that
the polyene antifungal antibiotic is gradually decomposed in an aqueous solution.
In the present invention, however, the polyene antifungal antibiotic is present in
oil droplets of the lipid and hence, are present in such a state that the antibiotic
is shielded from the surrounding environment. Therefore, enzymatic or non-enzymatic
decomposition can be prevented so that stability of the drug is also improved.
[0028] As described above, the fat emulsion particles in accordance with the present invention
are characterized by using the surface layer (for example, refined egg yolk lecithin)
in large quantities in its proportion to the nucleus of the emulsion particles (for
example, soybean oil), as compared to conventional fat emulsions using high calorie
supplementation fluid comprising soybean oil and egg yolk lecithin which belongs to
the prior art. By such a formulation, a stable fat emulsion of fine particles containing
the polyene antifungal antibiotic can be obtained for the first time.
[0029] In the fat emulsion of the present invention, it is necessary to use phospholipid
in an amount of 0.15 to 2 times that of the simple lipid.
[0030] By using the phospholipid in such an amount, the surface area at the part which becomes
the core of the emulsion particle by finely dividing the emulsion increases to cover
the core as the surface layer of the emulsion particles, whereby the amount of phospholipid
required to stabilize the emulsion can be sufficiently supplied.
[0031] Furthermore, this means that the amount of phospholipid required for stably retaining
the polyene antifungal antibiotic in the emulsion particles can be sufficiently supplied.
[0032] With the amount less than the lower limit described above, it is unavoidable that
coarse particles are intermingled so that any stable emulsion containing the drug
cannot be obtained. Where the phospholipid is used in an amount exceeding the upper
limit, it is unavoidable that liposome particles are intermingled so that any uniform
fat emulsion cannot be obtained.
[0033] It is required that the content of the polyene antifungal antibiotic in the present
invention be 5% (w/v) or less.
[0034] Examples of the simple lipid used in the present invention include neutral lipids
such as refined soybean oil, cotton seed oil, rape seed oil, sesame oil, corn oil,
peanut oil, safflower oil, triolein, trilinolein, tripalmitin, tristearin, trimyristin,
triarachidonin, etc. Additional examples also include sterol derivatives such as cholesteryl
oleate, cholesteryl linolate, cholesteryl myristate, cholesteryl palmitate, cholesteryl
arachidate, etc.
[0035] Neutral lipids are relatively easily decomposed by a variety of lipase present in
vascular endothelium, etc., whereas cholesterol derivatives undergo decomposition
by these enzymes only with difficulty so that stability further increases
in vivo. Therefore, the neutral lipids are preferable as the ingredients of the present invention.
[0036] Examples of the phospholipid which can be ued in the present invention include phospholipids
derived from egg yolk, soybean, bovine, swine, etc. and phospholipids obtained purely
synthetically or semi-synthetically. That is, examples include phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,
etc.
[0037] Additional examples include egg yolk phosphatidylcholine, soybean phosphatidylcholine
dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine,
dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, etc.
Hydrogenated products thereof may also be used. Among them, refined egg yolk lecithin
is a preferred representative.
[0038] In order to impart surface charge to the emulsion particles, lipids having a charge
such as stearylamine, dicetyl phosphate, phosphatidic acid, phosphatidylglycerol,
etc. may also be used.
[0039] In producing the fat emulsion of the present invention, various methods for producing
emulsions conventionally used heretofore may also apply as they stand. It is conventional
that for example, all of the ingredients including the drug are sufficiently finely
ground by using a pressure jet type homogenizer such as Manton-Gaulin type, etc.,
a microfluidizer, a ultrasonic homogenizer, etc. to form the fat emulsion of the present
invention.
[0040] In the production, sterols, fatty acids or derivatives thereof, which are generally
known as emulsifying aids or stabilizers and physiologically acceptable, may also
be incorporated. Representative examples of these substances include cholesterol,
oleic acid, etc.
[0041] The shape and particle diameter of the fat emulsion in accordance with the present
invention can be readily confirmed by an electron microscope, a particle diameter
analysis device of light scattering type, filtration through a membrane filter, etc.
[0042] In order to achieve a higher utilization value, other ingredients may also be added
to the fat emulsion of the present invention. As such ingredients, additives, auxiliary
substances and the like which are generally used for injection may be illustratively
shown.
For example, there are an antioxidant, a preservative, a stabilizer, an isotonic agent,
a buffering agent, and the like. The amounts required and the optimum amounts of these
additives, auxiliary substances, etc. may be varied depending upon purpose.
[0043] The thus obtained fat emulsion of the present invention is sterilized (for example,
sterilization by filtration, sterilization by high pressure steam, etc.), if necessary,
and sealed in an ampoule together with nitrogen gas. In addition, the fat emulsion
can be freeze-dried, if necessary. The freeze-dried fat emulsion of the present invention
can be thawed by adding a suitable solution thereto.
[0044] The fat emulsion of the present invention is generally administered intravenously
to human or various animals, for the purposes of treating or preventing antifungal
infections or viral infections. In this case, it is necessary to sufficiently control
the particle diameter, etc. of the emulsion particles.
[0045] In injection for intravenous application, it is known that when particles of 1 µm
or more are intermingled, various toxic conditions generally appear. The fat emulsion
of the present invention can be administered as injection intraarterially, intramuscularly,
intramedullarly and subcutaneously, as in conventional fat emulsions. In addition,
the fat emulsion of the present invention can be prepared into and used as eye drops,
nose drops, oral agents, inhalations, urinary-bladder injections, suppositories, ointments,
etc. Also in these cases, additives which are pharmaceutically acceptable bases, excipients,
etc. may also be added to the fat emulsion of the present invention.
[0046] A dose of the fat emulsion in accordance with the present invention which is administered
may be varied depending upon route for administration, preparation form, condition
and purpose but the dose of 1 to 1000 ml/time is generally sufficient as the emulsion.
A dose of the polyene antifungal antibiotic administered is in the range of 1 to 200
mg/time for adult.
[0047] As the polyene antifungal antibiotic which can be applied to the present invention,
there are Amphotericin B methyl ester, Nystatin, Trichomycin, Pimaricin, etc., in
addition to Amphotericin B.
[0048] According to the present invention, clinical utilization value of the polyene antifungal
antibiotic can be markedly enhanced. As the effects of the present invention, the
problems in the prior art are overcome and following effects are exhibited: (1) not
only hemolytic toxicity possessed by polyene antifungal antibiotics but also nephrotoxicity,
which is the serious problem to be solved, are markedly reduced; (2) migration of
drugs into the focus is improved; (3) uptake by the reticuloendothelial system is
prevented; (4) duration of blood concentration of drugs contained is realized; (5)
stability during storage is ensured; (6) production costs are reduced, etc. These
effects are obtained by the present invention for the first time.
[0049] The ingredients of the fat emulsion in accordance with the present invention are
mainly medically acceptable lipids which have been hitherto used practically as medical
drugs in clinical fields. Therefore, the fat emulsion of the present invention can
be used extremely safely.
BEST MODE FOR PRACTICING THE INVENTION
[0050] Hereinafter the present invention is described in more detail, with reference to
examples relating to the production of the fat emulsion in accordance with the present
invention. However, it is apparent that the present invention is not limited only
to these examples.
Preparation Example 1
[0051] After 3 mg of Amphotericin B, 0.5 g of refined soybean oil and 0.5 g of refined egg
yolk lecithin are mixed with each other and dissolved in 100 ml of chloroform/methanol
(1/1, v/v) mixture, the solvent is completely removed under reduced pressure using
a rotary evaporator. Thereto is added 8 ml of isotonic phosphate buffer solution.
The mixture is stirred with a homogenizer to form a coarse emulsion. Isotonic phosphate
buffer solution is added to the emulsion to make the volume 10 ml. Thereafter the
mixture is emulsified for 60 minutes under ice cooling, using a ultrasonic homogenizer
(Branson Model 185) to obtain a fine fat microemulsion containing Amphotericin B having
a mean particle diameter of 45 nm.
Preparation Example 2
[0052] After 3 g of Amphotericin B, 50 g of refined soybean oil and 15 g of refined egg
yolk lecithin are heated to about 60°C and mixed with each other, 500 ml of isotonic
phosphate buffer solution is added to the mixture to form a coarse emulsion. The coarse
emulsion is emulsified under high pressure using a Manton-Gaulin type homogenizer
to obtain a fine fat microemulsion containing Amphotericin B having a mean particle
diameter of 70 nm.
Preparation Example 3
[0053] After 30 mg of Amphotericin B, 0.6 g of refined soybean oil and 0.5 g of refined
egg yolk lecithin are mixed with each other and dissolved in 100 ml of chloroform/methanol
(1/1, v/v) mixture, the solvent is completely removed under reduced pressure using
a rotary evaporator. Thereto is added 8 ml of 0.24 M glycerine aqueous solution. The
mixture is stirred with a homogenizer to form a coarse emulsion. After 0.24 M glycerine
aqueous solution is added to the emulsion to make the volume 10 ml, the mixture is
emulsified for 60 minutes under ice cooling, using a ultrasonic homogenizer (Branson
Model 185) to obtain a fine fat microemulsion containing Amphotericin B having a mean
particle diameter of 93 nm.
Preparation Example 4
[0054] After 2 g of Amphotericin B, 20 g of refined soybean oil and 30 g of refined egg
yolk lecithin are heated to about 60°C and mixed with each other, 100 ml of 0.24 M
glycerine aqueous solution is added to the mixture. The mixture is stirred with a
homomixer to obtain a coarse emulsion. The coarse emulsion is emulsified under high
pressure using a microfluidizer to obtain a fine fat microemulsion containing Amphotericin
B having a mean particle diameter of 20 nm.
Preparation Example 5
[0055] After 1 mg of Amphotericin B, 0.5 g of cholesteryl oleate and 0.5 g of refined egg
yolk lecithin are mixed with each other and dissolved in 100 ml of chloroform/methanol
(1/1, v/v) mixture, the solvent is completely removed under reduced pressure using
a rotary evaporator. Thereto is added 8 ml of 0.24 M glycerine aqueous solution. The
mixture is stirred with a homogenizer to form a coarse emulsion. After 0.24 M glycerine
aqueous solution is added to the emulsion to make the volume 10 ml, the mixture is
emulsified for 60 minutes under ice cooling, using a ultrasonic homogenizer (Branson
Model 185) to obtain a fine fat microemulsion containing Amphotericin B having a mean
particle diameter of 55 nm.
Preparation Example 6
[0056] After 3 mg of Amphotericin B, 0.5 g of refined soybean oil and 0.1 g of dimyristoyl
phosphatidylglycerol are mixed with each other and dissolved in 100 ml of chloroform/methanol
(1/1, v/v) mixture, the solvent is completely removed under reduced pressure using
a rotary evaporator. Thereto is added 8 ml of 9% lactose aqueous solution. The mixture
is stirred with a homogenizer to form a coarse emulsion. After 9% lactose aqueous
solution is added to the emulsion to make the volume 10 ml, the mixture is emulsified
for 60 minutes under ice cooling, using a ultrasonic homogenizer (Branson Model 185)
to obtain a fine fat microemulsion containing Amphotericin B having a mean particle
diameter of 48 nm.
Preparation Example 7
[0057] After 3 mg of Amphotericin B, 0.5 g of refined soybean oil, 0.4 g of hydrogenated
egg yolk lecithin and 0.1 g of cholesterol are mixed with each other and dissolved
in 100 ml of chloroform/methanol (1/1, v/v) mixture, the solvent is completely removed
under reduced pressure using a rotary evaporator. Thereto is added 8 ml of 9% lactose
aqueous solution. The mixture is stirred with a homogenizer to form a coarse emulsion.
After 9% lactose aqueous solution is added to the emulsion to make the volume 10 ml,
the mixture is emulsified for 60 minutes under ice cooling, using a ultrasonic homogenizer
(Branson Model 185) to obtain a fine fat microemulsion containing Amphotericin B having
a mean particle diameter of 31 nm.
Preparation Example 8
[0058] After 0.5 g of albumin is added to the drug compositions containing Amphotericin
B obtained in Preparation Examples 1, 5 and 6, the composition is freeze-dried to
give the freeze-dried preparation.
[0059] The results obtained by evaluating the properties of the drug compositions containing
Amphotericin B in accordance with the present invention are shown below. In each test,
commercially available Amphotericin B preparation, various liposome preparations containing
Amphotericin B which belong to the prior art, and a conventional fat emulsion are
used for the purpose of comparison. Details of each sample are described below.
Sample 1: Drug composition containing Amphotericin B in accordance with the present
invention which is obtained in Preparation Example 1
Sample 2: Drug composition containing Amphotericin B in accordance with the present
invention which is obtained in Preparation Example 3
Comparative Sample 1: Commercially available Amphotericin B preparation for injection
(trademark: Fungizone (registered trademark), Squibb Japan)
Comparative Sample 2: Liposome preparation containing Amphotericin B which is prepared
according to Publication No. 1, composed of dimyristoyl phosphatidylcholine : dimyristoyl
phosphatidylglycerol = 7 : 3 in molar ratio and classified into multilamellar liposome
Comparative Sample 3: Liposome preparation containing Amphotericin B which is prepared
according to Publication No. 1, composed of dimyristoyl phosphatidylcholine : dimyristoyl
phosphatidylglycerol = 7 : 3 in molar ratio, obtained after ultrasonic treatment and
classified into small unilamellar liposome
Comparative Sample 4: Liposome preparation containing Amphotericin B which is prepared
according to Publication No. 1, composed of refined egg yolk lecithin, obtained after
ultrasonic treatment and classified into small unilamellar liposome
Comparative Sample 5: Liposome preparation containing Amphotericin B which is prepared
according to Publication No. 2, composed of refined soybean oil and refined egg yolk
lecithin
Test Example 1: Test on Hemolysis
[0060] Hemolytic action on purified rat erythrocyte was examined
in vitro with respect to Sample 1 and Comparative Sample 1. Comparative Sample 1 showed marked
hemolysis in an extremely low concentration (0.1 µg/ml or more) of Amphotericin B
but Sample 1 hardly showed even in the concentration of 200 times or more. It is thus
clear that the fat emulsions of the present invention greatly reduce hemolytic toxicity
possessed by Amphotericin B itself, as in conventionally known liposome preparations
and fat emulsion preparation.
Test Example 2: Acute Toxicity (in vivo)
[0061] Using ddY strain male mice (weighing about 20 g) as experimental animals, each of
Samples and Comparative Samples was intravenously administered from the tail vein
to evaluate their acute toxicity. Figs. 2 and 3 show survival rates of the mice 1
and 72 hours after single administration.
[0062] The survival rate evaluated 1 hour after the administration which is shown in Fig.
2 demonstrates toxic evaluation mainly due to hemolysis possessed by Amphotericin
B. Samples of the present invention tested all showed low toxicity. Among Comparative
Samples, reduction in toxicity due to hemolysis was noted in Comparative Samples 2
and 3. However, alleviation of acute toxicity was not noted in Comparative Samples
1, 4 and 5.
[0063] The survival rate evaluated 72 hours after the administration which is shown in Fig.
3 demonstrates toxic evaluation mainly due to nephrotic toxicity possessed by Amphotericin
B. Samples of the present invention tested all showed extremely low toxicity. However,
in all of Comparative Samples, toxicity appeared, indicating that nephrotoxicity is
remarkable as compared to Samples of the present invention.
[0064] It is evident that in the fat emulsions of the present invention, the effects of
reducing toxicity not only in the hemolytic toxicity observed immediately after administration
but also especially in the toxicity evaluated 72 hours after administration which
is considered to be due to nephrotoxicity are remarkable.
Test Example 3: Amount of Drug in Kidney (distribution into kidney)
[0065] Using SD strain male rats (weighing about 250 g) as experimental animals, each of
Samples of the present invention and Comparative Samples was intravenously administered
through the tail vein. The dose was 1 mg/kg as Amphotericin B. 18 hours after administration,
the kidney was removed and homogenized. Then the concentration of Amphotericin B in
kidney was determined by high performance liquid chromatography. The results are shown
in Table 1.
[0066] Where Samples of the present invention were administered, the Amphotericin B concentration
in kidney was less than the measurement limit in any case. Where Comparative Samples
were administered, however, Amphotericin B was detected in a high concentration in
all of the cases.
[0067] The fat emulsions of the present invention clearly achieve remarkable improvement
in distribution of the drug into kidney (lowering in migration), as compared to conventionally
known liposome preparations and fat emulsion preparation.
Table 1
Amount of Amphotericin B Distributed into Kidney |
|
Concentration in Kidney (µg/g) |
Sample 1 of This Invention |
Less than quantitative limit (<0.1) |
Sample 2 of This Invention |
Less than quantitative limit (<0.1) |
Comparative Sample 1 |
1.4 ± 0.1 |
Comparative Sample 2 |
1.3 ± 0.3 |
Comparative Sample 5 |
1.5 ± 0.4 |
(average value ± standard error, n = 3) |
Test Example 4: Evaluation of Kidney Function
[0068] Using SD strain male rats (weighing about 250 g) as experimental animals, each of
Samples of the present invention and Comparative Samples was intravenously administered
through the tail vein. The dose was 1 mg/kg as Amphotericin B and the drug was administered
every other 24 hours 3 times in total. 24 hours after the final administration, blood
was collected from the cervical vein to obtain serum. The amount of blood urea nitrogen
(BUN) used as an index of the kidney functions was determined using a commercially
available assay kit. The results are shown in Table 2. For control, physiological
saline was administered in the same manner and the thus obtained serum was used.
Table 2
Serological Evaluation of Kidney Function |
|
BUN (mg/dl) |
Control |
14.7 ± 1.7 |
Sample 1 of This Invention |
14.7 ± 1.2 |
Sample 2 of This Invention |
16.5 ± 1.5 |
Comparative Sample 1 |
29.2 ± 2.6 |
Comparative Sample 2 |
29.9 ± 2.1 |
Comparative Sample 5 |
37.8 ± 5.4 |
(average value ± standard error, n = 3) |
[0069] Where Samples of the present invention were administered, there was no difference
in BUN concentration between Samples and Control in any cases, indicating that no
disturbance was noted in kidney functions. However, where Comparative Samples were
administered, markedly high BUN concentration was noted in all cases, indicating that
disturbance in the kidney functions was noted. It is evident that in the fat emulsions
of the present invention, remarkable improvement in disturbance of kidney functions
can be achieved, as compared to conventionally known liposome preparations and fat
emulsion preparation.
Test Example 5: Change in blood concentration
[0070] Using SD strain male rats (weighing about 250 g) as experimental animals, each of
Samples of the present invention and Comparative Samples was intravenously administered
through the tail vein. The dose administered was 1 mg/kg as Amphotericin B. After
the administration, a small amount of blood was collected from the cervical vein to
obtain serum. The concentration of Amphotericin B in kidney was determined by high
performance liquid chromatography. The results are shown in Fig. 4.
[0071] Where Samples of the present invention were administered, the change of Amphotericin
B concentration in serum was higher in any cases than in Comparative Samples. On the
other hand, where any of Comparative Samples was administered, the concentration of
Amphotericin B in serum decreased. It is evident that the fat emulsions of the present
invention maintain remarkable blood concentration of Amphotericin B, as compared to
conventionally known liposome preparations and fat emulsion preparation.
Test Example 6: Distribution of Drug to Inflammatory Site
[0072] It is known that the site infected with fungi, etc. causes inflammation. Distribution
of the drug into experimentally induced inflammatory site was evaluated in the model
system.
[0073] Using SD strain male rats (weighing about 250 g) as experimental animals, 0.1 ml
of 2% λ-carrageenin was intrathoracically administered to cause experimental pleurisy.
Two hours and a half after, each of Samples of the present invention and Comparative
Samples was intravenously administered through the tail vein. The dose administered
was 1 mg/kg as Amphotericin B. After the administration, each animal was bled to death
from the abdominal aorta in each time to obtain the exudate in the thoracic cavity.
The concentration of Amphotericin B in the exudate was determined by high performance
liquid chromatography. The results are shown in Fig. 5.
[0074] It was shown that where any of Samples of the present invention was administered,
the change of Amphotericin B concentration in the exudate was higher than in any of
Comparative Samples. It is evident that the fat emulsions of the present invention
have the property of remarkably concentrating on the inflammatory site (infected site),
as compared to conventionally known liposome preparations and fat emulsion preparation,
and can achieve more effective and safer chemotherapy.
Test Example 7: Measurement of particle diameter
[0075] The particle diameters of Sample 1 and Sample 2 of the present invention were evaluated
using an apparatus for measuring dynamic light scattering particle diameter by laser
light. As the result, the particle diameter of Sample 1 of the present invention showed
about 20 to about 80 nm. Sample 1 did not contain particles having 1 µm or more. The
particle diameter of Sample 2 of the present invention showed about 70 to about 200
nm. Sample 2 did not contain particles having 1 µm or more.
[0076] It is evident that the fat emulsion of the present invention comprises extremely
fine and uniform emulsion particles. Furthermore the fat emulsion is free of particles
of 1 µm or more which are problematic when administered intravenously. It is therefore
evident that effective and safe chemotherapy can be attained.
Test Example 8: Antifungal test (in vitro)
[0077] Candida bacteria (C. Albicans) was cultured in Sabouraud's medium and each of Samples
of the present invention and Comparative Samples was added to the medium. The minimum
inhibitory concentration of Amphotericin B for inhibiting the growth of Candida was
determined to evaluate the antifungal activity of each sample. The results are shown
in Table 3. Each sample showed the antifungal activity in an extremely trace concentration
of Amphotericin B to inhibit the growth of Candida.
[0078] It was demonstrated that the fat emulsions of the present invention attain effective
and safe chemotherapy without adversely affecting the antifungal activity possessed
by Amphotericin B itself at all.
Table 3
Antifungal Activity (in vitro) |
|
Minimum Effective Concentration (µg/ml) |
Sample 1 of This Invention |
0.03 or less |
Sample 2 of This Invention |
0.16 or less |
Comparative Sample 1 |
0.20 or less |
Comparative Sample 3 |
0.14 or less |
BRIEF DESCRIPTION OF THE DRAWINGS
[0079] Fig. 1 shows the results of test of Sample 1 of the present invention and Comparative
Sample 1 on hemolysis
in vitro using rat erythrocyte. The abscissa designates concentration of Amphotericin B (µg/ml)
and the ordinate designates hemolytic ratio by %, wherein the curve obtained by connecting
black circles indicates Sample 1 of the present invention and the curve obtained by
connecting white circles indicates Comparative Sample 1.
[0080] Fig. 2 shows the results of evaluation of toxicity of each sample in terms of survival
rate of mice one hour after administration of each of Samples of the present invention
and Comparative Samples. The abscissa designates the dose when calculated as Amphotericin
B and the ordinate designates survival rate of mice one hour after the administration.
The respective samples are shown on the curve obtained by connecting black circles,
respectively.
[0081] Fig. 3 shows the results of evaluation of toxicity of each sample in terms of survival
rate of mice 72 hours after administration of each of Samples of the present invention
and Comparative Samples. The abscissa designates the dose when calculated as Amphotericin
B and the ordinate designates survival rate of mice 72 hours after the administration.
The respective samples are shown on the curve obtained by connecting black circles,
respectively.
[0082] Fig. 4 shows the change of concentration of Amphotericin B in serum after administration
of each of Samples of the present invention and Comparative Samples. The abscissa
indicates progress of time (hour) after administration of each sample and the ordinate
indicates concentration (µg/ml) of Amphotericin B in serum. The curves obtained by
connecting white circles, black circles, black squares, black triangles and white
triangles indicate the case of Sample 1 of the present invention, Sample 2 of the
present invention, Comparative Sample 1, Comparative Sample 2 and Comparative Sample
5, respectively.
[0083] Fig. 5 shows the change in concentration of Amphotericin B in the exudate of the
thoracic cavity in rats with experimentally induced pleurisy after administration
of each of Samples of the present invention and Comparative Samples. The abscissa
indicates progress of time (hour) after administration of each sample and the ordinate
indicates concentration (µg/ml) of Amphotericin B in the exudate. The curves obtained
by connecting white circles, black circles, black squares, black triangles and white
triangles indicate the case of Sample 1 of the present invention, Sample 2 of the
present invention, Comparative Sample 1, Comparative Sample 2 and Comparative Sample
5, respectively.
INDUSTRIAL APPLICABILITY
[0084] As described above, the fat emulsion in accordance with the present invention can
effect safe administration of polyene antifungal antibiotics such as Amphotericin
B, etc. in an effective dose, as a pharmaceutical preparation.